Characterization of yeast mutants lacking alkaline ceramidasesYPC1andYDC1

Abstract

Humans and yeast possess alkaline ceramidases located in the early secretory pathway. Single deletions of the highly homologous yeast alkaline ceramidases YPC1 and YDC1 have very little genetic interactions or phenotypes. Here, we performed chemical-genetic screens to find deletions/conditions that would alter the growth of ypc1∆ydc1∆ double mutants. These screens were essentially negative, demonstrating that ceramidase activity is not required for cell growth even under genetic stresses. A previously reported protein targeting defect of ypc1∆ could not be reproduced and reported abnormalities in sphingolipid biosynthesis detected by metabolic labeling do not alter the mass spectrometric lipid profile of ypc1∆ydc1∆ cells. Ceramides of ypc1∆ydc1∆ remained normal even in presence of aureobasidin A, an inhibitor of inositolphosphorylceramide synthase. Moreover, in caloric restriction conditions Ypc1p reduces chronological life span. A novel finding is that, when working backwards as a ceramide synthase in vivo, Ypc1p prefers C24 and C26 fatty acids as substrates, whereas it prefers C16:0, when solubilized in detergent and working in vitro. Therefore, its physiological activity may not only concern the minor ceramides containing C14 and C16. Intriguingly, so far the sole discernable benefit of conserving YPC1 for yeast resides with its ability to convey relative resistance toward H2O2.

Analysis of sphingolipids, sterols, and phospholipids in human pathogenic Cryptococcus strains

“…The experiments showed that the sphingolipid biosynthetic pathway is conserved in C. grubii (H99) and C. gattii (R265 and R272) strains. This is similar to the pathway that has been proposed in other fungi, such as C. albicans, A. fumigatus, and A. nidulans (34,37,(48)(49)(50)(51)(52)(53)(54). Sphingolipid biosynthesis can be divided into two parts with a common precursor, dhSph (d18:0).…”

“…Pathways of sphingolipid biosynthesis and genes involved therein are well-characterized in several fungi, such as S. cereviseae, Aspergillus fumigatus, Aspergillus nidulans, C. albicans, Pichia pastoris, and others (34,37,(47)(48)(49)(50)(51)(52)(53)(54). In one example of P. pastoris, the pathway of GlcCer biosynthesis very closely fits with the pathway present in Cryptococcus (19,21,54).…”

“…Further, both PhytoCer and OHPhytoCer structures can be converted to IPC structures by transfer of a phosphorylinositol group (23). Although the IPC structures and pathway of synthesis have been characterized in several fungi (36,(48)(49)(50)(51)(52)(53)(54), they remain poorly studied in Cryptococcus (23). In our MRM analysis (positive ion mode), we scanned for 28 IPC structures ( low-level IPC structures (IPC derivatives 2 and 3A).…”

The LUX Score: A Metric for Lipidome Homology

“…In future work, we will test how quantitative changes should be weighted with respect to structural changes. We will estimate such weight measures from well understood model systems based on larger data sets that are now becoming available (Voynova et al, 2014;Tarasov et al, 2014). However, this study shows that the structural composition of a lipidome is sufficient to recognize the degree of genetic alteration and growth temperature dependence in yeast strains in an unsupervised manner.…”

Saccharomyces cerevisiae Is Dependent on Vesicular Traffic between the Golgi Apparatus and the Vacuole When Inositolphosphorylceramide Synthase Aur1 Is Inactivated

“…Every 40 min the labeling media were replenished by adding 1 volume of 5-fold-concentrated SC medium (buffered as described above) to 4 volumes of culture. Labeling was terminated after 2 h, and lipids were extracted as recently described (24) and desalted using butanol-water partitioning or Folch partitioning; the latter does not completely retain M(IP) 2 C in the organic phase. Lipids were deacylated using NaOH as described previously (25).…”